Chemical, biological and immunological characterization of separated electrophoretic components of human chorionic...

Purified HCS preparations ofter show heterogeneity when assayed by electrophoresis, although they may be considered homogeneous according to other criteria (N-terminal amino acid analysis, gel filtration and ultracentrifugation patterns, etc.). Using DEAE-cellulose chromatography, the two main components corresponding to various degrees of hormone amidation were separated. The deamidation kinetic of the electrophoretically slower migrating component was studied and found to be sensitive to temperature and, to a lesser extent, to pH. Denaturing agents strongly increased the deamidation rate, while no effect was found by raising the ionic strength. No difference was evident between the two components and the parent hormone when compared (on a weight basis) in a specific HCS radioimmunoassay system. Even the precipitin reaction in agar gel with an anti-human growth hormone (HGH) serum did not show variations in cross-reactivity versus HGH. Moreover, groups of guinea pigs immunized with the single HCS components, gave antibodies reacting identically with the components and the parent HCS. The prolactin-like activity of the two components differed when determined by the pigeon crop-sac asay: the slower migrating component proved to be less active than the more deamidated one. On the other hand, the activity in two different radioreceptor assays (employing membranes from rabbit mammary gland and rat ventral prostate) was greater for the faster migrating component.